KR960005181B1 - Recombinant-htlv-ñ-protein and their usage - Google Patents

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Description

Recombinant HTLT-III Protein and Uses thereof

1 is a schematic of plasmid pREV 2.2, which is used to construct vectors that enhance novel proteins.

2 shows plasmid pREV 2.2 with multiple cloning sites.

3 schematically shows the HTLV-III envelope gene and novel recombinant proteins obtained therefrom.

The present invention relates to novel recombinant HTLT-III proteins and their use.

Human T-cell lymphotropic virus (HTLV-III), lymphadenopathy-associated virus (LAV), or AIDS-related retrovirus (ARV) has been found to be the cause of acquired immune deficiency syndrome (AIDS). See, Popovic, M., Sarngadharan, MG, Read, E. and Gallo RC, Science, 224: 497-500 (1984). The virus exhibits tropism towards the OKT ₄ ⁺ lymphocyte subset (see Klatzmann, D., Barre-Sinoussi, F., Nugeyre, MT, Dauguet, C., Vilmer E., Griscelli, C). , Brun-Vezinet, F., Rouzioux, C., Gluckman, JC, Chermann, JC and Montagnier, L., Science, 225: 59-63 (1984)].

Antibodies to serum of most AIDS and AIDS related complex (ARC) patients, and HTLV-III proteins in asymptomatic humans infected with viruses [Sarngadharan, MG, Popovic., M., Bruch, L., Schubach] , J. and Gallo, RC, Science, 224: 506-508 (1984), have enabled the development of immunologically based tests for detecting antibodies to these antigens. These tests are used to limit the spread of HTLV-III through transfusion by identifying blood samples of humans infected with the virus. Today's commercially available diagnostic tests use inactivated virus proteins as antigens.

In addition to allowing new approaches for diagnosis, recombinant DNA offers considerable potential for the development of vaccines for both bacteria and viruses (Wilsion, T., Bio / Technology, 2: 29-39). (1984)]. The organisms most widely used to express recombinant vaccines are E. coli. E. coli, S. cerevisiae and mammalian cell cultures. For example, subunit vaccines for foot and mouth disease (Kleid, DG, Yansura, D., Small, B., Dowbenko, D., Moore, DM, Brubman, MJ, McKercher, PD, m Morgan, DO, Robertson, BH, and Bachrach, HL, Science, 214: 1125-1129 (1981)] and vaccines against malaria [Young, JF, Hockmeyer, WT, Gross, M., Ripley Ballou, W ., Wirtz, RA, Trosper, JH, Beaudoin, RL, Hollingdale, MR, Miller, LM, Diggs, CL And Rosenberg, M., Science, 228: 958-962 (1985) have been synthesized in this coli. Another example is hepatitis B surface antigen produced by yeast [McAleer, W.J., Buynak, E.B., Maigetter, R.Z., Wampler, D.E., Miller, W.J. And Hilleman, MR, Nature, 307: 178-180 (1984)] and herpes vaccines produced in mammalian cells (Berman, PW, Gregory, T., Chase, D. and Lasky, LA, Science, 227: 1490-1492 (1984).

There is a real need for the development of vaccines against AIDS. However, it is not known that such a vaccine exists.

The present invention relates to novel recombinant HTLV-III proteins and their use. More particularly, the present invention relates to four novel recombinant HTLV-III outer membrane proteins that can be used for the diagnosis, prevention or treatment of AIDS. In addition, the recombinant HTLV-III outer membrane protein fragments of the present invention can be used to promote lymphocyte proliferative responses in humans infected with HTLV-III. This, in turn, stimulates the immune system to HTLV-III responses in such individuals, so that outer membrane protein fragments can provide protective action and can be an assessment of treatment.

These novel proteins are encoded on bacterial plasmids that can be used to transform appropriate hosts, such as this coli, using standard methods.

Expression vector plasmid pREV 2.2 is constructed from plasmid pBG1. A flowchart showing the construction of this plasmid is shown in FIG.

Plasmid pR 10 contains approximately 1275 bp of DNA encoding essentially the HTLV-III env gene from the Kpn I site to the Bg1II site. A suitable bacterial host, such as this plasmid of this coli, can be used to generate a recombinant recombinant HTLV-III 95 kD fusion protein (denoted R10). The amino acid sequence of the fusion protein R10 is shown in Table 8; DNA sequences encoding this protein are shown in Table 8A. Plasmid pPB1 essentially contains approximately 540 bp of DNA encoding the HTLV-III env gene from the PvuII site to the Bg1II site. This plasmid in a suitable host such as E. coli can be used to generate a recombinant recombinant HTLV-III 265 kD fusion protein (denoted PB1). The amino acid sequence of the fusion protein PB1 is shown in Table 9; DNA sequences encoding this protein are shown in Table 9A.

Plasmid p590 essentially contains approximately 1055 bp of DNA encoding the HTLV-III env gene from the PvuII site to the HindIII site. A suitable host, such as this plasmid of this collagen, can be used to generate a novel recombinant HTLV-III 86 kD protein (denoted 590). The amino acid sequence of fusion protein 590 is shown in Table 10; DNA sequences encoding this protein are shown in Table 10A.

Plasmid pKH1 essentially contains approximately 1830 bp of DNA encoding the HTLV-III evn gene from the Kpn I site to the HindIII site. This plasmid in a suitable host such as this coli can be used to generate a novel recombinant HTLV-III 70 kD protein (denoted KH1). The amino acid sequence of the fusion protein KH1 is shown in Table 11; DNA sequences encoding this protein are shown in Table 11A.

Plasmid pBG1 in E. coli host MS 371 was prepared by the Northern Regional Research Laboratory (NRRL), U.S. Deposited with the Department of Agriculture, Peoria, IIIinois, USA. It is permanently deposited. This Coli MS 371 (pBG1), NRRL B-15904 was deposited on November 1, 1984. This Coli MS 371, NRRL B-15129 is currently available to the public. The coli SG 20251, NRRL B-15918, was deposited on 12 December 1984. NRRL B-15904 and NRRL B-15918 will be available to the public if the patent describing them is granted. Other cultures deposited with NRRL and their deposit dates and access numbers are as follows.

The deposit was deposited on February 3, 1988, with the Korean spawn association under accession numbers KFCC-10514, 10515, 10516, 10517 and 10518, respectively.

The deposits will last for at least 30 years at the NRRL Depository and will be made available to the public if a patent describing them is granted. The deposit is also available as complies with foreign patent law in the country to which the counterpart of this patent application, or its application is filed. It should be noted, however, that the availability of the deposit does not constitute a patent right to enforce the invention in the event of a compromise of the patent rights granted by the government's decision.

The novel HTLV-III proteins of the invention can be shaped in these organisms using plasmids containing inducible galactose promoters from Saccharomyces cerevisiae. See Broach, JR, Li, Y., Wu, LC And Jayaram, M., in, Experimental Manipulation of Gene Expression, P. 83, ed, M. Inouye, Academic Press (1983). These plasmids are also called YEp51 and YEp52. See Broach, J.R. et al. (1983)], E. coli clone, gene for β-lactamase, yeast LEU2 gene, 2 μm replica and 2 μm cyclic REP3 locus. Recombinant gene expression is driven by the yeast GAL 10 gene promoter.

Galactose and alcohol dehydrogenases [Bennetzen J.L. And Hall, B. D., J. Biol. Chem., 257: 3018 (1982); Ammerer, G. in Methods in Enzymology, Vol, 101, p. 192 (1988)], phosphoglycerate kinases [Derynck, R., Hitzeman, R.A., Gray, P.W., Goeddel, D.V. on Experimental Manipulation of Gene Expression, p. 247, ed. M. Inouye, Academic Press (1983). Triose phosphate isomerase [Alber, T. and kawasaki, G., J. Molec. and Applied Genet., 1: 419 (1982)], or enolase [Innes, M.A. et al., Science, 226: 21 (1985)] can be used.

The genes described herein can be expressed in monkey cells. Genes encoding these proteins are described in Okayama, H. and Berg, P., Molec. and Cell Biol., 3: 280 (1983) and one of the plasmids described in this document, or when cloned into COS cells transformed with these plasmids, the synthesis of HTLV-III protein is immunologically Can be detected.

Other mammalian cell gene expression / protein production systems can also be used. Examples of other such systems are vaccinia or virus expression systems [Moss, B., Immunology Today, 6: 24 (1985); Chakrabarti, S., Brechling, K., Moss, B., Molec. and Cell Biol., 5: 3403 (1985)] and vectors derived from rat retroviruses (Mulligan, R.C., in Experimental Manipulation of Gene Expression, P. 155, ed. M. Inouye, Academic Press (1983).

The HTLV-III proteins of the present invention are described in solid phase peptide synthesis techniques such as BOC and FMOC [Merrifield, R. B., J. Amer. Chem. Soc., 85: 2149 (1963); Chang, C. and Meienhofer. J., Int. J. Peptide Protein Res., 11: 246 (1978)].

As is well known in the art, the amino acid sequence of a protein is determined by the nucleotide sequence of DNA. Different nucleotide sequences can encode specific amino acids because of the redundant genetic code, ie, one or more coding nucleotide triplets (codons) can be used for most of the amino acids used to synthesize the protein. That is, the genetic code can be written as follows.

Phenylalanine (Phe) TTK

Leucine (Leu) XTY

Isoleucine (Ile) ATM

Methionine (Met) ATG

Val QTL

Serin QRS

Proline (Pro) CCL

Threonine (Thr) ACL

Alanine (Gla) GCL

Tyrosine (Tur) TAK

Histidine CAK

Glutamine (Gln) CAJ

Asparagine (Asn) AAK

Lys AAJ

Aspartic Acid (Asp) GAK

Glutamic Acid (Glu) CAJ

Cysteine TGK

Tryptophan (Trp) TGG

Arginine (Arg) WGZ

Glycine GGL

Termination Signal TAJ

Termination Signal TGA

Description: Each 3-letter deoxynucleotide triplet corresponds to a trinucleotide of mRNA having a 5'-end on the left and a 3'-end on the right. All DNA sequences described herein are sequences of the backbone corresponding to the mRNA sequence that replaced uracil with thymine. Letters represent purine or pyrimidine bases that form deoxynucleotide sequences.

A is adenine, G is guanine, C is cytosine, T is thymine, X is T or C if Y is A or G, X is C if Y is C or T, and X is C If Y is A, G, C or T, if X is T then Y is A or G, if Z is A or G, W is C or A, if Z is C or T, W is C, Z is A, G, C or T if W is C, Z is A or G if W is A, QR is TC if S is A, G, C or T, and S is T or C Where QR is AG, J is A or G, K is T or C, L is A, T, C or G and M is A, C or T.

The above indicates that novel amino acid sequences of the HTLV-III proteins of the present invention can also be prepared by nucleotide sequences other than those described herein. Novel amino acid sequences of these HTLV-III proteins, or functionally equivalent nucleotide sequences encoding HTLV-III antigens or immunogens or fragments thereof having therapeutic activity, can also be prepared by known synthetic methods. Accordingly, the present invention also encompasses such functionally equivalent nucleotide sequences.

Thus, the scope of the present invention encompasses not only the specific nucleotide sequence described herein, but also all of the equivalent nucleotide sequences encoding substantially the same HTLV-III antigen or immunogen or molecule having therapeutic activity.

In addition, the scope of the present invention is intended to include not only the specific amino acid sequences described, but also similar sequences encoding protein fragments having the comparable ability to induce the formation of certain HTLV-III antibodies and / or to bind antibodies. .

The term “equivaient” is practically performed as a nucleotide sequence identified in the present invention that conventional patent practice yields molecules having substantially the same HTLV-III antigen, immunogen or therapeutic activity in a host of substantially the same kind. Is used to mean a nucleotide sequence. Within this definition are included HTLV-III antigens or immunogens or sub fragments having therapeutic activity.

As described above, nucleotide sequences encoding the HTLV-III antigens or immunogens or therapeutic activity of the invention within the scope of the field of genetic engineering can be used to generate HTLV-III proteins through microbiological processes. Methods of fusing a sequence into an expression vector and transforming or transfecting a host, such as a eukaryotic cell (yeast or mammalian cell) or a prokaryotic cell (bacterial cell), include other well known proteins such as insulin, interferon, It is a standard method used to produce human growth hormone, IL-1, IL-2, etc., HTLV-III protein by microbiological means or tissue-culture technology in accordance with the present invention using a similar method or a clear modification thereof. It may also be prepared.

Nucleotide sequences described herein can be prepared using "gene maching" by methods well known in the art. This has been made possible by the technical literature on nucleotide sequences.

The restriction enzymes described can be purchased from Bethesda Research Laboratories, Gaithersburg, MD or New England Biolabs, Beverly, MA. Use the enzyme according to the instructions provided by the supplier.

Various methods used to prepare plasmids and to transform host organisms are well known in the art. These methods are all described in the following references: Maniatis, T., Fritsch, EF, and Sambrook, J., Molecular Cloniong: A Laboratory Manual., Cold Spring Harbor Laboratory, New York (1982). Within the scope of engineering expertise, DNA can be extracted from microbial cells, subjected to restriction enzyme digestion, electrophoresis of DNA fragments, terminal insertion and annealing of plasmids, or DNA linkages, Cells, such as E. coli cells, can be transformed, plasmid DNA can be prepared, proteins can be electrophoresed or DNA can be sequenced.

Immunological assays using the HTLV-III protein of the present invention can take a variety of forms. Preferred types are solid phase immunoassay assays. In this type of assay, HTLV-III protein is immobilized on a solid phase to form an antigen-immunoresorbent. Immunosorbents are incubated with the sample to be tested. After incubation for an appropriate time, the immunosorbent is separated from the sample and then labeled anti-human IgG antibody is used to detect human anti-HTLV-III antibody bound to the immunosorbent. The amount of label bound to the immunosorbent can be compared to the positive and negative controls to assess the presence of anti-HTLV-III antibodies.

Immunosorbents can be prepared by adsorbing or coupling purified HTLV-III protein onto a solid phase. Various solid phases can be used, such as beads made of glass, polystyrene, polypropylene, dextran or other substrates. Other suitable solid phases include tubes or plates formed from or coated with these materials.

HTLV-III proteins can be covalently or non-covalently incorporated into the solid phase using techniques such as covalent bonds via amide or ester bonds or adsorption. After attaching the HTLV-III protein to the solid phase, the solid phase can be post-coated with animal protein, for example 3% fish gelatin. This provides a blocking protein that reduces nonspecific adsorption of the protein to the immunosorbent surface.

The immunosorbent is then incubated with the sample to be tested for anti-HTLV-III antibodies. In blood screening, plasma or serum is used. Plasma or serum is diluted with plasma or serum from normal animals. Diluted plasma or serum is derived from the same animal species that is the source of anti- (human IgG) antibody. Preferred anti- (human IgG) antibodies are goat anti- (human IgG) antibodies. In other words, in a preferred embodiment, the diluent is the serum or plasma of goat.

Culture conditions, ie pH temperature, and incubation time are not critical. These variables can be optimized by routine experimentation. In general, the cultivation is performed at pH 7-8 at about 45 ° C. in buffer for 1-2 hours.

After incubation, the immunosorbent and sample are separated. Separation may be carried out by conventional separation techniques such as precipitation or centrifugation. The immunosorbent is then removed from the sample to remove the inhibitor.

Immunosorbents are incubated with labeled anti- (human IgG) antibodies (tracers) to detect human antibodies bound thereto. Immunosorbents are generally incubated with a solution of labeled anti- (human IgG) antibody containing serum or plasma from a small amount (about 1%) of animal species provided as a source of anti- (human IgG) antibody. Anti- (human IgG) antibodies can be obtained from any animal source. However, goat's anti- (human IgG) antibodies are preferred. The anti- (human IgG) antibody may be an antibody against an FC fragment of human IgG, eg, an anti- (human IgG) FC antibody of goat.

The anti- (human IgG) antibody or anti- (human IgG) FC antibody may be labeled with a radioactive material such as ¹²⁵ I; May be labeled with an optical label such as a fluorescent material; Or an enzyme such as horseradish peroxidase. Anti-human antibodies can also be biotinylated and avidin labeled, and then used to detect their binding to an immunosorbent.

After incubation with the labeled antibody, the immunosorbent is separated from the solution and the label bound to the immunosorbent is measured. Depending on the choice of label, the measurement can be performed in various ways. If the label is a radio gamma emitter, the label can be detected with a gamma counter, or if the label is a fluorescent material, the label can be detected by fluorescence measurement. For enzymes, label detection can be performed colorimetrically using a substrate for the enzyme.

The amount of label bound to the immunosorbent is compared to the positive and negative controls to determine the presence of anti-HTLV-III antibody. Control tests are generally performed incidentally with the sample to be tested. Positive control is type blue containing antibody against HTLV-III; Negative controls are serum from healthy individuals that do not contain antibodies to HTLV-III.

For convenience and standards, reagents for performing immunoassay assays can be collected in assay kits. Kits for blood screening include, for example, (a) polystyrene beads coated with an immunosorbent such as HTLV-III protein; (b) diluents for serum or plasma samples, eg serum or plasma from normal goats; (c) anti- (human IgG) antibodies, eg, goat's anti- (human IgG) antibodies in a buffered aqueous solution containing about 1% goat serum or plasma; And (d) serum containing an antibody against a positive control, eg, at least one novel HTLV-III protein; And (e) sera from a healthy individual that does not contain an antibody against a negative control, eg, at least one novel HTLV-III protein.

If the label is an enzyme, additional components of the kit may be substrates for the enzyme.

Another form of assay for anti-HTLV-III antibodies is an antigen sandwich assay. In this assay, labeled HTLV-III proteins are used instead of anti- (human IgG) antibodies to detect anti-HTLV-III antibodies bound to immunosorbents. This assay is based on the bivalency of antibody molecules. One binding site of the antibody binds to the antigen attached to the solid phase, and the second site enables binding of the labeled antigen. The assay procedure is substantially the same as described above for the immunoassay assay, except that after incubation with the sample, the immunosorbent is incubated with a solution of labeled HTLV-III protein. HTLV-III proteins can be labeled with radioisotopes, enzymes, and the like for this type of assay.

In a third form, the bacterial protein binding to the FC fragment of the IgG molecule, Protein A, as a labeled tracer can be used to detect anti-HTLV-III antibodies conjugated to an immunosorbent without disrupting antigen-antibody interactions. , Protein A can be easily labeled with radioisotopes, enzymes or other detectable species.

Immunological assays using HTLV-III protein have several advantages over assays using whole (or disrupted) viruses. Assays based on the HTLV-III protein alleviate the concern of significant increases in infectious virus and inherent survival activity associated with cell culture and virus production. In addition, the assay will help mitigate actual or perceived aspects of adverse reactions to AIDS by technicians working in hospital clinics and blood banks performing the test.

Vaccines of the HTLV-III proteins described herein, and variants thereof having antigenic properties, can be prepared by methods well known in the art. For example, such vaccines may be prepared as injectable solutions, for example liquid solutions or suspensions. Solid forms, ie liquids, in solutions or suspensions may be prepared before injection. Optionally, the formulation may be emulsified. The active antigenic component or ingredients can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol and the like, and mixtures thereof. In addition, if desired, the vaccine may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, or adjuvants that enhance the effectiveness of the vaccine. Vaccines may be administered parenterally or, for example, by subcutaneous or intramuscular injection. Additional formulations suitable for other dosage forms include suppository glycols or triglycerides. Suppositories can be formulated from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably in the range of 1% to about 2%. Oral formulations may include commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, economics, pills, capsules, sustained release preparations or powders and may contain about 10% to about 95%, preferably about 25% to about 70%, of the active ingredient.

Proteins can be formulated into vaccines in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts with the free amino group of the peptide and acid addition salts with inorganic acids (eg hydrochloric acid or phosphoric acid) or organic acids (eg acetic acid, oxylic acid, tartaric acid, mandelic acid, etc.). . Salts with free carboxyl groups are also derived from inorganic bases (e.g. sodium, potassium, ammonium, calcium or iron hydroxide) and organic bases (e.g. isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, etc.). Can be induced.

The vaccine is administered in a therapeutically effective and immunogenic amount in a manner suitable for dosage formulation. The amount to be administered depends on the subject being treated, the ability of the immune system of the subject to synthesize antibodies, and the degree of protection desired. The exact amount of active ingredient required for treatment is administered at the discretion of the attending physician and is unique to each individual. However, a suitable dosage range can be about several hundred micrograms of active ingredient per individual. Suitable regimens for initial administration and booster shots may also vary, but subsequent injections or other administrations are generally given at one or two week intervals after the initial administration.

HTLV-III has been found to occur in amino acid sequence changes, particularly in the outer membrane genes. Starcich, B.R,., Cell, 45: 637-648 (1986): Hahn, B.H. et al., Science, 232: 1548-1553 (1986). More than 100 variants are analyzed by molecular cloning and restriction enzyme recognition assays, some of which are nucleotide sequence analyzed. Some of these are described in RF (Popovic, M. et al., Science, 224: 497-500 (1984)), WMJ-1 [Hahn, BH.H. et al., Science, 232: 1548-1553 (1986)], LAV [Wain-Hobson, S. et al., cell, 40: 9-17 (1985)], and ARV-2 [Sánchez] Pescador, R. et al., Science, 227: 484-492 (1985), are known HTLV-III isolates. Although the present invention describes sequences from one HTLV-III isolate, the appropriate outer membrane region of any HTLV-III isolate can be prepared using the manner described herein for the preparation of R10, PB1, 590, and KH 1. Can produce. HTLV-III proteins from different viral isolates can be used in vaccine formulations as described above to protect against infection by different HTLV-III isolates. In addition, vaccine formulations are prepared using one or more recombinant sulfurogenic proteins from one or more HTLV-III isolates to provide immunity and thus provide greater protection against AIDS.

The following examples illustrate the method of the present invention including the preferred embodiments. These examples should not be understood as being limited. All solvent mixture proportions are by volume unless otherwise indicated.

Example 1

Construction of Plasmid pREV 2.2

Plasmid pREV 2.2 expression vector is constructed from plasmid pBG1. Plasmid pBG1 can be isolated from its E. coli host by well known methods, for example using clarified lysate-equivalent density methods. Like pBG1, pREV 2.2 expresses the inserted gene behind the E. coli promoter. The differences between pBG1 and pREV 2.2 are as follows:

1. pREV 2.2 lacks the functional replication power of the plasmid (rop) protein.

2. pREV 2.2 has a trp A electronic terminator inserted at the Aat II site. This sequence ensures transcription termination of overexpressed genes.

3. pREV 2.2 has genes that provide resistance to ampicillin and chloramphenicol, whereas pBG1 only provides resistance to ampicillin.

4. pREV 2.2 contains sequence coding sites for several restriction endonucleases.

The following procedure (shown in Figure 1) is used to impart each of the four changes described above.

1a. 5 μg of plasmid pBG1 is limited to Nde I to yield two fragments of approximately 2160 and 3440 bp.

1b. After inactivation of Nde I, 0.1 μg of DNA from the degradation mixture was subjected to an intermolecular linking reaction (200 μl of reaction volume using standard T4 ligase [New England Biolabs, Beverly, MA] reaction conditions). Treated with T4 DNA ligase. Intramolecular linkage of the 3440 bp fragment yields an ampicillin resistant plasmid. The ligation mixture is used to transform the receptor, strain E. coli JM 103 (available from New England Biolabs) and to select ampicillin resistant clones by standard methods.

1c. The resulting plasmid pBG1 ΔN, with 2160 bp Nde I fragments deleted from pBG1, was selected by measuring restriction distribution patterns with Nde I and Sal I, which produced plasmids from ampicillin resistant clones (approximately 1790 and 1650 bp). Product fragments). This deletion inactivates the rop gene, which controls plasmid replication.

2a. Then 5 μg of pBG1 ΔN were digested with Eco R I and BCl I and then a larger fragment, approximately 2455 bp fragment, was isolated.

2b. Synthetic double-stranded fragments are prepared with the structures shown in Table 1 in the manner described in the literature: Itakura, K., Rossi, J. J. and Wallace, R. B. Ann. Rev. Biochem., 53: 323-356 (1984), and references cited therein]. This fragment has Bcl I and Eco R I adhesion ends and contains the recognition sequences for several restriction endonucleases.

2c. 0.1 μg of 2455 bp Eco R I-Bcl I fragment and 0.01 μg of synthetic fragment are ligated using T4 DNA ligase, and the ligation mixture is used to transform competent cells of strain JM103. . Cells containing the recombinant plasmid, wherein the synthetic fragment is inserted in pBG1 ΔN between the Bcl I site and the Eco R I site, are selected by digesting the plasmid with Hpa I and Eco R I. Diagnostic fragment sizes are approximately 2355 and 200 bp. This plasmid is called pREV 1.

2d. 5 μg of pREV 1 is digested with Aat II and uniquely cleaved.

2e. Synthesize the double stranded fragment shown in Table 2: This fragment has an Aat II adhesion terminus and contains a trp A electron termination sequence.

2f. 0.1 μg AatII digested pREV 1 is linked with 0.01 μg synthetic fragment using T4 DNA ligase in 2 μl volume.

2 g. Competent cells of strain JM103 are transformed and empicillin resistant clones are selected.

2h. Cells containing the correct construct are isolated using Kpn I, EcoRI double restriction digests of plasmids isolated from selected colonies. The fragments generated from Kpn I, EcoRI are approximately 2475 and 80 bp in size. This flask is called pREV 1 TT and contains a trpA transcription terminator.

3a. 5 μg of pREV 1TT prepared as described above (by standard method) is cleaved with Nde I and Xmn I and approximately 850 bp fragment is isolated.

3b. 5 μg of plasmid pBR 325 (BRL, Gaithersburg, MD), containing genes that confer resistance to ampicillin and tepracycline as well as chloramphenicol, was cleaved with Bcl I and the ends were clenopolymerase and deoxynucleotides. Use to smooth. After inactivation of the enzyme, the mixture is treated with Nde I and approximately 3185 bp fragments are isolated. This fragment contains chloramphenicol and ampicillyl resistance genes and replicators.

3c. 0.1 μg of Nde I-Xmn fragment from pREV1 TT and Nde I-Bcl I fragment from pBR 325 were linked in 20 μl with T4 DNA ligase and the mixture was used to isolate competent cells of strain JM 103. Transform. Cells that are resistant to both ampicillin and chloramphenicol are selected.

3d. Using EcoRI and Nde I double digests of plasmids from selected clones, the plasmids are screened to yield approximately 2480, 1145 and 410 bp fragments. This is called the plasmid pREV1TT / ch1 and has a gene that is resistant to both ampicillin and chloramphenicol.

4a. The double stranded fragments shown in Table 3 are synthesized. This fragment with smooth ends and Sst I adhesion ends contains the recognition sequences for several restriction enzyme sites.

4b. 5 μg of pREV1TT / ch1 is cleaved into Nru I (cutting about 20 nucleotides from the Bcl I site) and Sst I (cutting in multiple cloning sites). A larger fragment, approximately 3990 bp fragment, is separated from the agarose gel.

4c. 0.1 μg of Nru I-Sst I fragment and 0.01 μg of synthetic fragment from pREV1TT / ch1 are treated with T4 DNA ligase in a volume of 20 μl.

4d. This mixture is used to transform strain JM103 and to select empicillin resistant clones.

4e. The plasmid is purified from several clones and then screened by digestion with Mlu I or Cla I. Recombinant clones with novel multiple cloning sites, when digested with one of these enzymes, each cleave the plasmid only once, thus providing one fragment.

4f. Identify the sequence of multiple cloning sites. This is done by limiting the plasmid to Hpa I and PvuII and separating 1395 bp fragments, then cloning it into the Sma I site of mp 18 and sequencing with dideoxynucleotide sequencing using standard methods.

4 g. This plasmid is called pREV 2.2 and is shown in FIG.

Example 2

Construction and Expression of pR10

A plasmid that contains approximately 1275 bp of DNA encoding substantially the HTLV-III env gene from the Kpn I site to the Bg1II site, from which it can synthesize an approximately 95 kD fusion protein containing this portion of the gp 120 outer membrane protein. pR10 can be constructed as follows:

1. Synthesis of DNA having the sequence shown in Table 4: This DNA fragment can be synthesized by standard methods [Itakura, et al., Supra, and references cited therein] and part of gp 120 Encrypt It has a blunt end on the 5 'end and an end connected to the BamHI overhang on the 3' end.

2. Restrict 5 μg of plasmid pBG1 to Bcl I, overhang the prominent end with Clenow polymerase and deoxyribonucleotide triphosphate (d NTP _S ) and limit this fragment to Bam HI and then the large fragment, Approximately 8.9 Kd fragment is separated from the agarose gel.

3. 0.1 μg of the fragment shown in Table 4 was linked to 0.31 μg of pBG 1 fragment using T4 DNA ligase in a volume of 20 μl and using this mixture, competent cell strain SG 20251 [Gottesman, S ., Halpern, E. and Trisler, P., Journal of Bacteriology, 148: 265-273 (1981)], followed by selection of ampicillin resistant transformants.

4. Using the AhaIII restriction pattern of the purified plasmid, the cells containing the recombinant plasmid with the synthetic fragments oriented by linking the blunt ends of the fragments to the pBG 1 fragment, filling the Bcl I termini and then linking the BamHI overhanging ends together Screening. The appropriate plasmid is digested with AhaIII to give fragments approximately 5300, 3170, 690, 640, 330 and 20 bp in length.

5. Strains containing this recombinant plasmid were treated with 2% medium [2% yeast extract, bactotryptone, casamino acid (Difco, Detroit, MI), 0.2% monobasic potassium, 0.2 containing 50 μg / ml ampicillin. When grown in% dibasic potassium and 0.2% dibasic sodium] and the entire supplement of cellular protein is electrophoresed on an SDS-polyacrylamide gel, approximately 95 kD of major proteins are stained with coomassie blue, or Or by Western blot analysis using sera selected from AIDS, ARC, and HTLV-III infected individuals as probes.

Example 3

Purification of Recombinant Protein Containing HTLV-III Outer Membrane Sequence from Plasmid pR10

1. Growth of Cells:

Cells are grown in 2% medium in a 10 L volume of Chemap fermentor (Chemapec, Woodbury, NY). Fermentation temperature is 37 ℃, pH is 6.8, air is injected at a rate of 1vvm. Plasmid selection is performed using 50 μg / ml ampicillin. Typical cell yield (wet weight) is 30 g / l.

2. Cell Lysis:

50 g (wet weight) of E. coli cells containing the recombinant HTLV-III outer fusion protein were collected in 50 mM Tris-Cl pH 8.0, 5 mM potassium ethylenediaminetetraacetic acid (KEDTA), 5 mM dithiothreitol (DTT), 15 mM β-mer Resuspend in 100 mL (final volume) of buffer consisting of captoethanol, 0.5% Triton X-100 and 5 mM phenylmethylsulfonylfluoride (PMSF). 300 mg of lysozyme is added and the suspension is incubated for 30 minutes at room temperature.

This material is dissolved using BEAD-BEATER ^™ (Bicspec Products, Bartlesville, OK) containing the same volume of 0.1 to 0.15 μm glass beads. Dissolution is carried out for 6 minutes at room temperature at 1 minute intervals. The liquid is separated from the beads and centrifuged at 20,000 × g for 2.5 hours. The supernatant is removed and the pellet is dissolved in 100 mL buffer consisting of 8M urea, 230 mM Tris-Cl pH 0.8, 5 mM DTT, 15 mM β-mercaptoethanol, 5 mM PMSF and 1 mM KEDTA. The pellet is solubilized using a polytron homogenizer (Beckman, Berkely, Calif.) And then centrifuged at 20,000 × g for 2 hours.

3. Diethylaminoethyl (DEAE) Chromatography

The supernatant was filled with DEAE Fast Flow Sepharose (Pharmacia, Piscataway, NJ) equilibrated in a buffer consisting of 8M urea, 20 mM Tris-Cl pH 8.0, 15 mM β-mercaptoethanol and 1 mM KEDTA at room temperature. Load on a 550 ml column (5 cm x 28 cm). The column is washed with 1.5 L equilibration buffer and the protein is eluted with a 5.0 L linear gradient of 0-0.8 M NaCl in equilibration buffer. HTLV-III protein is eluted in 0.2 M NaCl and assayed by SDS-polyacrylamide electrophoresis to give approximately 95 kD of major protein.

Fractions containing HTLV-III protein were collected and the protein was purified using a pressurized positive cell concentrator (Amicon, Danvers, MA) with a 10,000 MW cut-off membrane (YM-10, Amicon) attached. Concentrate on. The concentrate was packed into a 500 ml column (2.5 cm × 100 cm) filled with ultrafine Sephacryl S-300 (Pharmacia) equilibrated in a buffer consisting of 8 M urea, 20 mM Tris-Cl pH 8.0, 15 mM β-mercaptoethanol and 1 mM KEDTA. Load on). The column is eluted with equilibration buffer at room temperature. It is maintained at a flow rate of 0.5 ml. HTLV-III protein elutes at approximately 40% of the column volume.

Example 4

Construction and Expression of Plasmid pPB1

The plasmid pPB1, which contains approximately 540 bp of DNA encoding substantially the HTLV-IIIenv gene from the PvuII site to the BgIII site, from which the approximately 26 kD fusion protein containing this portion of the gp120 outer membrane protein can be synthesized is It can be constructed as follows.

1. Synthesize DNA having the sequence shown in Table 5: This DNA fragment can be synthesized by standard methods and encodes a portion of gp120. It has a blunt end on the 5 'end and an end connected to the BamHI overhang on the 3' end.

2. Limit 5 μg of plasmid pREV 2.2 to EcoRV and BamHI and separate the larger fragment, approximately 4 kD fragment, from the agarose gel.

3. Link to 0.1 μg of pREV 2.2 fragment using T4 DNA ligase with a volume of 20 μg of fragment 0.1 μg of fragment shown in Table 5, and transform competent cell strain SG 20251 using the ligation mixture, Ampicillin resistant transformants are selected.

4. Using the AhaIII restriction pattern of the purified plasmid, select cells containing recombinant plasmids with the synthetic fragments oriented by linking the blunt ends of the fragments to the pREV 2.2 EcoRV ends and the BamHI overhanging ends together. The appropriate plasmid is digested with AhaIII to give fragments approximately 1210, 1020, 750,690,500,340 and 20 bp in length. Strains containing this recombinant plasmid were grown in 2% medium containing 50 μg / ml ampicillin and the entire supplement of cellular proteins was electrophoresed on an SDS-polyacrylamide gel and approximately 26 kD of protein was transformed into Coomassie Blue. Stains can be visualized by staining or by Western blot analysis using serum as a selected probe from AIDS, ARC, or HTLV-III infected individuals.

Example 5

Purification of Recombinant Protein Containing HTLV-III Outer Membrane Sequence from Plasmid pPB1

1. Growth of Cells:

Cells are grown in 2% media in 10 L volume of KemaM fermentor. Fermentation temperature is 37 ℃, pH is 6.8, air is injected at a rate of 1vvm. Plasmid selection is performed using 50 μg / ml ampicillin and 20 μg / ml chloramphenylcol. Typical cell yield (wet weight) is 30 g / l.

2. Cytolysis

50 g (wet weight) of E. coli cells containing the recombinant HTLV-III outer membrane fusion protein were transferred to 50 mM Tris-Cl pH 8.0, 5 mM KEDTA, 5 mM DTT, 15 mM β-mercaptoethanol, 0.5% Triton X-100 and 5 mM PMSF Resuspend in 100 ml (final solution) of buffer. 300 mg of lysozyme is added and the suspension is incubated for 30 minutes at room temperature.

This material is dissolved using BEAD-BEATER ^™ (Biospec Products, Bartlesville, OK) containing the same volume of 0.1 to 0.15 μm glass beads. Dissolution is carried out for 6 minutes at room temperature at 1 minute intervals. The liquid is separated from the beads and centrifuged at 20,000 × g for 2.5 hours. The supernatant is removed and the pellet is resuspended in 100 mL buffer consisting of 6M guanidine-hydrochloride, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM β-mercaptoethanol, 5 mM PMSF and 1 mM KEDTA.

The pellet is solubilized using a polytron homogenizer and then centrifuged at 20,000 × g for 2 hours.

The supernatant (90 mL) is dialyzed against 4 L of buffer consisting of 8M urea, 20 mM Tris-Cl, pH 8.0 1 mM DETA and 15 mM β-mercaptoethanol. Dialysis is performed for 8 hours or more each time with 3 exchanges of buffer. Spectraphor dialysis tubes (S / P, McGraw Park, IL) with 3.5 kD cut-off are used.

3. CM Chromatography

550 ml (5 cm × 28) packed with CM fast flow Sepharose (Pharmacia) equilibrated in dialysis in a buffer consisting of 8M urea, 10 mM potassium phosphate pH 7.0, 15 mM β-mercaptoethanol and 1 mM KEDTA at room temperature Cm). The column is washed with 2 L equilibration buffer and the protein is eluted with a 5.0 L linear gradient of 0 to 0.4 M NaCl. Assay by SDS-polyacrylamide gel electrophoresis resulted in HTLV-III protein (26 kD) eluting at approximately 0.2 M Nacl.

Example 6

Construction and Expression of Plasmid p590

The plasmid p590, which contains approximately 1055 bp of DNA encoding substantially the HTLV-III env gene from the PvuII site to the HindIII site, from which the approximately 86 kD fusion protein containing this portion of the gp160 outer membrane protein can be synthesized It can be constructed as follows:

1. Synthesize the DNA having the sequence shown in Table 6: This DNA fragment can be synthesized by standard methods and encodes a portion of gp160. It has a blunt end on the 5 'end and an end that connects to the HindIII overhang on the 3' end.

2. Limit 5 μg of plasmid pREV 2.2 to EcoRV and HindIII, then separate the larger fragment, approximately 4 kD fragment, from the agarose gel.

3. Link 0.1 μg of the fragment shown in Table 6 to a 0.1 μg pREV 2.2 fragment using T4 DNA ligase in a volume of 20 ml and transform the competent cell strain SG20251 using the ligation mixture, followed by ampi Cylin resistant transformants are selected.

4. Using the AhaIII restriction pattern of the purified plasmids, select cells containing recombinant plasmids with the synthetic fragments oriented by linking the smooth ends of the fragments to the pREV 2.2 EocRV ends and the HindIII overhanging ends together. Appropriate plasmids are digested with AhaIII to yield fragments of approximately 1740,1020,750,690,500,340 and 20 lengths.

5. 5 μg purified plasmid from this strain is limited to Nde I and Sma I. Approximately 1425 bp fragments are separated from the agarose gel. A 1505 bp fragment is fused to the DNA encoding the fragment of gp160.

6. 5 μg of pBG 101 is limited to Bam HI and the protruding ends are filled with Klenow polymerase and dNTP _S and then Nde I. Approximately 6.5 kD fragments are separated from the agarose gel.

7. Link 0.1 μg of Nde I-Sma I fragment with 0.1 μg of pBG1 fragment using T4 DNA ligase, transform competent cell strain SG 20251 using a linkage mixture, and then transform into ampicillin resistant Screen sieves.

8. Using the AhaIII restriction pantone of the purified plasmid, connect the smooth Sma I end of the fragment to the end filled with Bam HI and link the Nde I overhangs together to connect the cells containing the recombinant plasmid with the synthetic fragments oriented. Select. The appropriate plasmid is digested with AhaIII to give fragments approximately 5900, 1020, 690, 430 and 20 bp in length.

9. If a strain containing this recombinant plasmid is grown in 2% medium containing 50 μg / ml ampicillin and the entire supplement of cellular protein is electrophoresed on an SDS-polyacrylamide gel, approximately 86 kD of protein is subjected to Cooma It can be visualized by staining with blue, or by western blot analysis using serum selected from AIDS, ARC, or HTLV-III infected individuals as a probe.

Example 7

Purification of Recombinant Protein Containing HTLV-III Outer Membrane Sequence from Plasmid p590

1. Growth of Cells

Cells are grown in 2% medium in a 10 L volume of Kemaph fermentor. The fermentation temperature is 37 ° C., pH is 6.8 and air is injected at a rate of 1 vvm. Plasmid selection is performed using 50 μg / ml ampicillin. Typical cell yield (wet) weight is 30 g / l.

2. Cell Lysis

50 g (wet weight) of E. coli cells containing the recombinant HTLV-III outer membrane fusion protein were transferred to 50 mM Tris-Cl pH 8.0, 5 mM KEDTA, 5 mM DTT, 15 mM β-mercaptoethanol, 0.5% Triton X-100 and 5 mM PMSF. Resuspend in 100 ml (final volume) of buffer. Add 300 mg of lysozyme and incubate the suspension for 30 minutes at room temperature.

This material is dissolved using Bead-Beater ^™ containing 0.1 to 0.15 mm glass beads. Dissolution is carried out for 6 minutes at room temperature at 1 minute intervals. The liquid is separated from the beads and centrifuged at 20,000 × g for 2.5 hours. The supernatant is removed and the pellet is resuspended in 100 mL buffer consisting of 6M guanidine-hydrochloride, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM β-mercaptoethanol, 5 mM PMSF and 1 mM KEDTA. The pellet is solubilized using a polytron homogenizer and then centrifuged at 20,000 × g for 2 hours.

The supernatant (90 mL) is dialyzed against 4 L of buffer consisting of 8 M urea, 20 mM Tris-Cl pH 8.0 1 mM EDTA and 15 mM β-mercaptoethanol. Dialysis is performed for 8 hours or more each time with 3 exchanges of buffer. Spectropodia dialysis tubes with 3.5 kD MW cut-off are used.

3. Diethylaminoethyl (DEAE) Chromatography

550 mL column (5 cm × 5) packed with DEAE Fast Flow Sepharose (Pharmacia) equilibrated in a buffer consisting of 8M urea, 20 mM Tris-Cl pH 80 15 mM β-mercaptoethanol and 1 mM KEDTA at room temperature 28 cm). The column is washed with 1.5 L equilibration buffer and the protein is eluted with a 5.0 L linear gradient of 0-0.8 M NaCl in equilibration buffer. The HTLV-III protein is eluted at 0.4 M NaCl and assayed by SDS-polyacrylamide electrophoresis to give a protein of approximately 86 kD predominantly.

Fractions containing HTLV-III protein are pooled and the protein is concentrated to 10 ml using a pressurized cell positive pressure concentrator (Amicon) with 10,000 MW cut-off membrane (YM-10, Amicon). The concentrate was packed into a 500 ml column (2.5 cm x 100 cm) filled with ultra-fine Sephacryl S-300 (Pharmacia) equilibrated in a buffer consisting of 8 M urea, 20 mM Tris-Cl pH 8.0 15 mM β-mercapto ethanol and 1 mM KEDTA. Load it to the yarn. The column is eluted with equilibration buffer at room temperature. Maintain a flow rate of 0.5 ml / min. HTLV-III protein elutes at about 40% of the column volume.

Example 8

Construction and Expression of Plasmid pKH1

A plasmid pKH1 that contains approximately 1830 bp of DNA encoding substantially the HTLV-III env gene from the Kpn I site to the HindIII site, from which it can synthesize an approximately 70 kD fusion protein containing this portion of the gp160 outer membrane protein. Can be constructed as follows:

1. Synthesize DNA having the sequence shown in Table 7: This DNA fragment can be synthesized by standard methods and encodes a portion of gp160. It has a blunt end on the 5 'end and an end that connects to the HindIII overhang on the 3' end.

2. Limit 5 μg of plasmid pREV 2.2 to Mlu I, treat DNA with Klenow polymerase to smooth the ends, then treat with HindIII and separate a larger fragment, approximately 5 kD, from agarose gel do.

3. 0.1 μg of the fragment shown in Table 7 was linked to 0.1 μg of pREV 2.2 fragment using T4 DNA ligase in a volume of 20 μl, and the competent cell strain CAG 629 was transformed using the ligation mixture, followed by The silin resistant transformants are selected.

4. Using the AhaIII restriction pattern of the purified plasmid, select cells containing recombinant plasmids with the synthetic fragments oriented by linking the blunt ends of the fragments to the pREV 2.2 Mlu I fragments and the HindIII overhanging ends together. The appropriate plasmid is digested with AhaIII to give fragments approximately 1730, 1020, 750, 690, 640, 600, 340 and 20 bp in length. When a strain containing this recombinant plasmid is grown in 2% medium containing 50 μg / ml ampicillin and the entire supplement of cellular proteins is electrophoresed on an SDS-polyacrylamide gel, approximately 70 kD of protein is coomassie blue. Staining or by Western blot analysis using serum as a probe selected from AIDS, ARC, or HTLV-III infected individuals.

Example 9

Purification of Recombinant Protein Containing HTLV-III Outer Membrane Sequence from Plasmid pKH1

1. Growth of Cells

Cells are grown in 2% media in 10 L volume of KemaM fermentor. The fermentation temperature is 32 ° C., pH is 6.8 and air is injected at a rate of 1 vvm. Plasmid selection is performed using 50 μg / ml ampicillin. Typical cell yield (wet weight) is 30 g / l.

2. Cell Lysis

50 g of E. coli cells containing the recombinant HTLV-III outer membrane fusion protein (wet weight) were transferred to 50 mM Tris-Cl pH 8.0, 5 mM KEDTA, 5 mM dithiothreitol (DTT), 15 mM β-mercaptoethanol, 0.5% Triton X Resuspend in 100 mL (final volume) of buffer consisting of -100 and 5 mM PMSF. Add 300 mg of lysozyme and incubate the suspension for 30 minutes at room temperature.

This material is dissolved using BEAD-BEATERF ^™ (Biospec Products) containing the same volume of 0.1 to 0.15 μm glass beads. Dissolution is carried out for 6 minutes at room temperature at 1 minute intervals. The liquid is separated from the beads and centrifuged at 20,000 × g for 2.5 hours. The supernatant is removed and the pellet is dissolved in 100 ml buffer consisting of 8M urea, 20 mM Tris-Cl pH 8.0 5 mM DTT, 15 mM β-mercaptoethanol, 5 mM PMSF and 1 mM KEDTA. Pellets are solubilized using a polytron homogenizer (Beckman, Berkeley, Calif.) And then centrifuged at 20,000 × g for 2 hours.

3. DEAE Chromatography

Supernatant, 550 mL column (5 cm × 5) packed with DEAE Fast Flow Sepharose (Pharmacia) equilibrated in buffer consisting of 8M urea, 20 mM Tris0Cl pH8.0, 15 mM β-mercaptoethanol and 1 mM KEDTA at room temperature. 28 cm). The column is washed with 1.5 L equilibration buffer and the protein is eluted with a 5.0 L linear gradient of 0-0.8 M NaCl in equilibration buffer. HTLV-III protein is eluted in 0.2 M NaCl and assayed by SDS-polyacrylamide electrophoresis to give approximately 70 kD of protein.

Fractions containing HTLV-III protein are pooled and the protein is concentrated to 10 ml using a pressurized cell positive pressure concentrator (Amicon) with 10,000 MW cut-off membrane (YM-10, Amicon). The concentrate was packed into a 500 ml column (2.5 cm × 100 cm) filled with ultrafine Sephacryl S-300 (Pharmacia) equilibrated in a buffer consisting of 8 M urea, 20 mM Tris-Cl pH 8.0, 15 mM β-mercaptoethanol and 1 mM KEDTA. Load on). The column is eluted with equilibration buffer at room temperature. Maintain a flow rate of 0.5 ml / min. HTLV-III protein elutes at approximately 40% of the column volume.

4. SDS-Polyacrylamide Electrophoresis

Fractions containing KH 1 are pooled and the protein is concentrated using a pressurized cell positive pressure concentrator with a 10,000 MW cut-off membrane. 2 mg of protein was mixed with loading buffer and described in M.W. Hunkapiller, E. Lujan, F. Ostrander, and L.E. Hood, Methods in Enzymology, 91: 227-236 (1983), are electrophoresed via preparative SDS-polyacrylamide gels (40 cm × 20 cm × 4 mm). 70 kD HTLV-III protein is visualized by staining with Coomassie Blue and then eluted from the gel as described above. Proteins can be recovered from SDS by precipitation with acetone. Dynan, W.J., Jendrisak, J.J., Hager, D.A. And Burgess, R. R., J. Biol. Chem., 256: 5860-5865 (1981).

TABLE 1

TABLE 2

TABLE 3

TABLE 4

TABLE 5

TABLE 6

TABLE 7

TABLE 8

Amino acid sequence of the fusion protein R10

TABLE 8A

Nucleotide sequence encoding the fusion protein R10

TABLE 9

Amino acid sequence of the fusion protein PB1

TABLE 9A

Nucleotide sequence encoding the fusion protein PB1

TABLE 10

Amino acid sequence of fusion protein 590

TABLE 10A

Nucleotide Sequence Encoding Fusion Protein 590

TABLE 11

Amino acid sequence of the fusion protein KH1

TABLE 11A

Nucleotide sequence encoding the fusion protein KH1

Claims (42)

A recombinant DNA transfer vector comprising DNA having all or a portion of the following nucleotide sequence or equivalent nucleotide sequence containing a base encoding a HTLV-III envelope protein fragment designated R10.

A recombinant DNA transfer vector comprising DNA having all or a portion of the following nucleotide sequence or equivalent nucleotide sequence containing a base encoding a HTLV-III outer membrane protein fragment designated PB1.

A recombinant DNA transfer vector comprising DNA having all or part of the following nucleotide sequence or equivalent nucleotide sequence containing a base encoding a HTLV-III outer membrane protein fragment designated 590.

The DNA transfer vector of claim 1, wherein the DNA transfer vector has been transferred to and replicated in a eukaryotic or prokaryotic host. 3. The DNA transfer vector of claim 2, wherein the DNA transfer vector is transferred to and replicated in a eukaryotic or prokaryotic host. 4. The DNA transfer vector according to claim 3, which is transferred to and replicated in a eukaryotic or prokaryotic host. A host transformed with the transfer vector of claim 1. A host transformed with the transfer vector of claim 2. A host transformed with the transfer vector of claim 3. HTLV-III outer membrane protein fragment having the following amino acid sequence and designated R10, or a mutant thereof.

HTLV-III outer membrane protein fragment having the following amino acid sequence and designated PB1, or a mutant thereof.

An HTLV-III outer membrane protein fragment having the following amino acid sequence and designated 590, or a mutant thereof.

A plasmid selected from plasmid pREV1, plasmid pPEV1TT, plasmid pREV1TT / ch1, plasmid pREV2.2, plasmid pR10, plasmid pPB1 and plasmid p590. The method of claim 13, wherein the plasmid pREV2.2. The plasmid pR10 according to claim 13. The plasmid pPB1 of claim 13. 14. The plasmid p590 of claim 13, wherein the plasmid p590. DNA having the following nucleotide sequence or an equivalent nucleotide sequence that contains a base that encodes an HTLV-III outer membrane protein fragment designated R10.

DNA having the following nucleotide sequence or an equivalent nucleotide sequence that contains a base that encodes an HTLV-III outer membrane protein fragment designated as PB1.

DNA having the following nucleotide sequence or an equivalent nucleotide sequence that contains a base encoding a HTLV-III outer membrane protein fragment wherein the region to be translated is designated 590.

An immunochemical assay that detects or quantifies antibodies against HTLV-III in fluid using HTLV-III protein selected from R10, PB1 and 590. The immunochemical assay of claim 21, wherein the HTLV-III protein is R10. The immunochemical assay of claim 21, wherein the HTLV-III protein is PB1. The immunochemical assay of claim 21 wherein the HTLV-III protein is 590. (A) An immunosorbent comprising a solid phase with an HTLV-III protein selected from R10, PB1, and 59-590, together with a sample of the biological fluid to be tested, the anti-HTLV-III antibody in the sample is added to the immunosorbent. Incubation under conditions that allow binding, and (b) separating the immunosorbent from the sample; (c) determining whether the antibody is bound to an immunosorbent as an indication of anti-HTLV-III in the sample, wherein the antibody against HTLV-III in the biological fluid is detected. The method of claim 25, wherein determining whether the antibody is bound to an immunosorbent comprises: culturing the immunosorbent with an antibody labeled against an antigen of a species from which the biological fluid is derived; After culturing, the immunosorbent is separated from the labeled antibody; Detecting a label associated with an immunosorbent. The method of claim 25, wherein determining whether the antibody is bound to an immunosorbent comprises: incubating the immunosorbent with a labeled HTLV-III protein selected from R10, PB1, and 590; Separating the immunosorbent from the labeled HTLV-III protein; Detecting a label associated with an immunosorbent. The method of claim 25, wherein determining whether the antibody is bound to the immunosorbent comprises: incubating the immunosorbent with labeled Protein A; Separating an immunosorbent from labeled Protein A; Detecting a label associated with an immunosorbent. (a) providing an immunosorbent comprising beads coated with HTLV-III protein selected from RIO, PBI and 590; (b) incubating the immunosorbent with the serum or plasma sample under conditions such that the anti-HTLV-III antibody in the sample can be combined with the immunosorbent; (c) separating the immunosorbent from the sample; (d) incubating the immunosorbent with labeled anti- (human IgG) antibody under conditions that allow the anti- (human IgG) antibody to bind to human anti-HTLV-III antibodies bound to the immunosorbent; (e) separating the immunosorbent from the unbound anti-human IgG antibody; (f) measuring the label bound to the immunosorbent as an indication of the presence of the antibody against HTLV-III in the sample, wherein the antibody against HTLV-III in human serum or plasma samples is measured. How to detect. 30. The method of claim 29, wherein the immunosorbent further comprises a post-coat of animal protein. The method of claim 29, wherein the labeled anti- (human IgG) antibody is an animal antibody and the serum or plasma sample is diluted with normal serum of the same animal species. 32. The method of claim 31, wherein the anti- (human IgG) antibody is a goat antibody and the serum or plasma sample is diluted with normal goat serum. The method of claim 29, wherein the anti- (human IgG) antibody is labeled with a radioisotope, enzyme, or fluorescent compound. An immunosorbent comprising a solid phase with an HTLV-III protein selected from R10, PB1 and 590 for use in solid phase immunochemical assays of antibodies against HTLV-III. 35. The immunosorbent of claim 34, wherein the solid phase is glass or plastic beads, wells of microtiter plates or test tubes. 35. The immunosorbent of claim 34, further comprising a post-coat of animal protein. A vaccine composition comprising an HTLV-III protein or proteins having antigenic properties of R10, PB1 or 590 in a pharmaceutically acceptable vehicle. The vaccine composition of claim 37, wherein the HTLV-III protein has the antigenic properties of R10. 38. The vaccine composition of claim 37, wherein the HTLV-III protein has the antigenic properties of PB1. The vaccine composition of claim 37, wherein the HTLV-III protein has an antigenic property of 590. (a) A sample of a biological fluid to be tested with a solid phase to which an HTLV-III protein selected from R10, PB1 and 590 is attached, which is under conditions that allow the anti-HTLV-III antibody in the sample to bind an immunosorbent. Immunosorbents comprising; (b) labeled HTLV-III antibodies; And (c) means for detecting a label that binds to an immunosorbent. 16. A kit for use in detecting an antibody against HTLV-III in a biological fluid. 42. The kit of claim 41, wherein the anti-HTLV-III antibody is labeled with an anti- (human IgG) antibody which is a detectable label.

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